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At least 559 records · Page 31Linked to original sources
PubMed2001

Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein.

Flock house virus (FHV), a member of the family Nodaviridae, is a nonenveloped, icosahedral insect virus whose capsids are assembled from 180 copies of a single type of coat protein. The viral genome is split between two segments of single-stranded positive-sense RNA, RNA1 and RNA2, which are packaged into a single virion. We previously demonstrated that synthesis of FHV coat protein in the baculovirus expression system results in assembly of virus-like particles whose capsids are indistinguishable from those of native virions, although the encapsidated RNA represents primarily cellular RNA. In contrast, expression of a deletion mutant lacking N-terminal residues 2-31 results in formation of multiple types of particles which differ in size, shape, and RNA contents. We postulated that the polymorphism was imposed by the type of RNA that the coat protein selected for packaging. In the current study we tested this hypothesis by analyzing the assembly of the mutant coat protein in Drosophila cells in the presence of replicating FHV RNAs. As anticipated, the resulting particles had the same shape and dimensions as wt virions. Surprisingly, however, they contained little RNA2 while packaging of RNA1 was not affected. Small amounts of defective interfering RNAs, which emerged rapidly in the presence of the mutant coat protein, were also detected. Taken together, these observations confirm our earlier hypothesis that selection of nonviral RNAs for packaging can significantly alter the assembly process. In addition, they demonstrate that the N-terminus of the FHV coat protein contains important determinants for recognition and packaging of RNA2. Our results provide the first evidence that encapsidation of the two genomic RNAs occurs independently and that the coat protein uses different regions for the recognition of RNA1 and RNA2.

Amino Acid Sequence↗
PubMed1983

Persistent baculovirus infections: Spodoptera frugiperda NPV and Autographa californica NPV in Spodoptera frugiperda cells.

Establishment of a persistent infection of Spodoptera frugiperda nuclear polyhedrosis virus (NPV) in Spodoptera frugiperda (S.f.) cells occurred in three phases: the first phase was characterised by high levels of cell infection and death, the second phase by decreasing cell infection levels leading to the final phase where less than one per cent of the cells were infected during any subculture. The virus persisted at this level of infection provided the cells were maintained by regular subculturing and incubated at the optimum growth temperature of 27 degrees C. Because of the low proportion of cells infected, cultures of virus-free cells could be selected ('cured') by dilution of the persistent infection without the use of viral antiserum. Unlike the parent S.f. cells, cultures of cured cells were partially resistant to infection with S. frugiperda NPV or infection with an unrelated baculovirus Autographa californica NPV. A. californica NPV, which is cytolytic for the parent S.f. cell line, established a persistent infection in the cured cells. The establishment pattern was similar to that previously found for S. frugiperda NPV and only one to five per cent of the cells were infected at equilibrium. Cured cells from the A. californica NPV persistent infection were highly resistant to infection with both S. frugiperda NPV and A. californica NPV. All attempts to find a viral interference phenomenon to explain the resistance of the cured cells were unsuccessful. All cell types adsorbed virus equally well. Slower growth of S.f. cells cured from the persistent A. californica NPV infection is the only difference so far observed between any of the S.f. cell types.

Animals↗
PubMed1979

Non-occluded particles of nuclear polyhedrosis virus infecting Galleria mellonella L.: titration in vivo and in vitro. Brief report.

Infectivity of non-occluded particles of nuclear polyhedrosis virus infecting Galleria mellonella L. was determined with bioassay technique in vivo and with plaque assay method in vitro using SCLd 135 (Quiot) established cell culture. A plaque-forming virus unit corresponds to about 50 ID50 for Galleria larvae injected into hemocoel for non-cloned and 0.1-1.0 ID50 for a virus isolate cloned from an infective plaque.

Animals↗
PubMed1985

Isolation of large and small plaque variants of the Autographa californica nuclear polyhedrosis virus. Brief report.

Variants of the Autographa californica nuclear polyhedrosis virus which produce large (1.4 mm) or small (0.5 mm) plaques on Spodoptera littoralis cells have been isolated. Yields of extracellular virus and polyhedra by the large plaque variant were six-fold higher and 140-fold lower, respectively, than those obtained with the small plaque variant. However the two variants could not be distinguished when Spodoptera frugiperda cells were used.

Animals↗
PubMed1981

Plaque assay of Heliothis zea baculovirus employing a mixed agarose overlay.

The nuclear polyhedrosis virus of Heliothis zea has been titrated in Heliothis zea cells by the plaque method, using 1 percent mixed agarose containing a mixture of Seakem and Ultra pure agarose. Visible plaques, formed 8 days postinfection, ranged in diameter from 0.5 to 2 mm. Dose-response experiments indicated that a single particle initiated the formation of a plaque. The titration of Heliothis zea baculovirus by the newly described plaque method provides an accurate technique for the determination of virus concentration.

Animals↗
PubMed2001

Temperature-dependent expression of a squid Kv1 channel in Sf9 cells and functional comparison with the native delayed rectifier.

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.

Animals↗
PubMed1996

Cytotoxicity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide.

Neem seed preparations contain not only azadirachtin as the active insect antifeedant or growth regulator but also a variety of their limonoids, some of which are cytotoxic to N1E-115 neuroblastoma (mouse), 143B.TK- osteosarcoma (human) and Sf9 (insect) cultured cell lines. The most potent of these limonoids is nimbolide with an IC50 ranging from 4 to 10 microM, and averaging 6 microM for the three cell lines. Other limonoids of decreasing potency and their average IC50 values (microM) are epoxyazadiradione 27 microM, salannin 112 microM, and nimbin, deacetylnimbin and azadirachtin each >200 microM (practically nontoxic). Nimbolide at 10 microM acts rapidly in the neuroblastoma cells to induce blebbing associated with disruption of plasma membranes almost instantaneously and 50% loss of cell viability with 30 min. At 5 microM nimbolide, the cells become elongated and assume a neuronal shape accompanied by spikes and lamellipodia within 1-2 hr followed shortly thereafter by extensive cytological changes and and vacuolization associated with irreversible processess leading to cell death. Calcium is apparently not involved the cytotoxic effect since a calcium-free medium, leading to profound morpholigical changes, does not alter the sensitivity to nimbolide. In contrast, epoxyazadiradione requires higher concentrations and a few hr for 50 % viability loss without major morphological changes, indicating a difference in mode of action for nimbolide and epoxyazadiradione. and epoxyazadiradione.

Animals↗
PubMed2000

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis.

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.

Amino Acid Sequence↗
PubMed1995

Kinetic properties of the alpha 2 beta 1 and alpha 2 beta 2 isozymes of the Na,K-ATPase.

The presence of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in most mammalian tissues has hindered the understanding of the roles of the individual isoforms in directing Na,K-ATPase function. Expression of the Na,K-ATPase subunits in insect cells using recombinant baculoviruses has proven to be a useful system for the study of Na,K-ATPase function. Using this system, we have expressed the rat Na,K-ATPase alpha 2 beta 1 and alpha 2 beta 2 isoforms in Sf-9 insect cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. Both beta 1 and beta 2 isoforms can efficiently assemble with the alpha 2 subunit to produce catalytically competent Na,K-ATPase molecules. The analysis of the kinetic properties of both isozymes showed that alpha 2 beta 1 and alpha 2 beta 2 have equivalent sensitivities to ouabain, and similar turnover numbers and apparent affinities for K+ and ATP. The dependence on Na+, however, differs between the isozymes, with alpha 2 beta 2 displaying a slightly higher apparent affinity for the cation than alpha 2 beta 1. In addition, the even greater kinetic differences between Na,K-ATPase isozymes varying in alpha isoforms may be important in further differentiating the enzyme. Thus, when compared to the rat alpha 1 beta 1 Na,K-ATPase expressed in Sf-9 cells, the alpha 2 beta 1 and alpha 2 beta 2 isozymes have a lower apparent affinity for K+ and a higher affinity for Na+ and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Glycosylation of shaker potassium channel protein in insect cell culture and in Xenopus oocytes.

We have studied the glycosylation of Shaker K+ channel protein made in two expression systems: an insect cell culture line and amphibian oocytes. In both systems, two potential sites for N-linked glycosylation were modified. The modified sites were located between the first and second putative transmembrane segments, S1 and S2. Although the same sites appeared to be glycosylated in both systems, the fraction of protein glycosylated and the size, structure, or composition of the oligosaccharide chains added were quite different. The results indicate that the S1-S2 loop is extracellular, consistent with a cytoplasmic location for the N-terminus and a transmembrane disposition for hydrophobic segment S1. We have also shown that glycosylation occurs in two stages in oocytes, generating an immature and a mature form of Shaker protein. However, glycosylation is not required either for the assembly of functional channels or for their transport to the cell surface.

Amino Acid Sequence↗
PubMed1995

Two distinct functions of the carboxyl-terminal tail domain of NF-M upon neurofilament assembly: cross-bridge formation and longitudinal elongation of filaments.

Neurofilaments are the major cytoskeletal elements in the axon that take highly ordered structures composed of parallel arrays of 10-nm filaments linked to each other with frequent cross-bridges, and they are believed to maintain a highly polarized neuronal cell shape. Here we report the function of rat NF-M in this characteristic neurofilament assembly. Transfection experiments were done in an insect Sf9 cell line lacking endogenous intermediate filaments. NF-L and NF-M coassemble to form bundles of 10-nm filaments packed in a parallel manner with frequent cross-bridges resembling the neurofilament domains in the axon when expressed together in Sf9 cells. Considering the fact that the expression of either NF-L or NF-M alone in these cells results in neither formation of any ordered network of 10-nm filaments nor cross-bridge structures, NF-M plays a crucial role in this parallel filament assembly. In the case of NF-H the carboxyl-tail domain has been shown to constitute the cross-bridge structures. The similarity in molecular architecture between NF-M and NF-H suggests that the carboxyl-terminal tail domain of NF-M also constitutes cross-bridges. To examine this and to further investigate the function of the carboxyl-terminal tail domain of NF-M, we made various deletion mutants that lacked part of their tail domains, and we expressed these with NF-L. From this deletion mutant analysis, we conclude that the carboxyl-terminal tail domain of NF-M has two distinct functions. First, it is the structural component of cross-bridges, and these cross-bridges serve to control the spacing between core filaments. Second, the portion of the carboxyl-terminal tail domain of NF-M that is directly involved in cross-bridge formation affects the core filament assembly by helping them to elongate longitudinally so that they become straight.

Amino Acid Sequence↗
PubMed2002

Sp1 and AP2 regulate but do not constitute TATA-less human TAF(II)55 core promoter activity.

Human TAF(II)55 (hTAF(II)55), a component of the general transcription factor TFIID, is the only general transcription factor encoded by an intronless gene identified thus far. Analysis of the TATA-less hTAF(II)55 promoter-proximal sequence reveals putative binding sites for STAT-1, MEF2, E2F, Sp1, AP2, AREB6 and E47. Using chromatin immunoprecipitation, DNase I footprinting and electrophoretic mobility shift assays, we demonstrate that Sp1 and AP2 can bind simultaneously to juxtaposed Sp1- and AP2-binding sites in the hTAF(II)55 promoter-proximal region and functionally modulate hTAF(II)55 promoter activity, as evidenced by reporter gene assays performed in transiently transfected human C-33A and insect SL2 cell lines. Interestingly, removal of all the promoter-proximal Sp1-binding sites does not impair the function of the hTAF(II)55 core promoter. Moreover, a 52-bp DNA fragment containing only the hTAF(II)55 initiator (Inr) and downstream promoter element (DPE) is able to support Gal4-VP16-mediated activation in vivo and in vitro. Our data suggest that Sp1, although it plays an enhancing role in hTAF(II)55 gene expression, is not essential for hTAF(II)55 core promoter activity. Interestingly, mutations introduced at the Inr and DPE differentially affect the selection of transcription start sites, suggesting that these two core promoter elements play a non-redundant role in the function of TATA-less promoters.

Base Sequence↗
PubMed2001

Analysis of the capsid processing strategy of Thosea asigna virus using baculovirus expression of virus-like particles.

Thosea asigna virus (TaV), a putative member of the genus Betatetravirus of the family Tetraviridae, is predicted to have a novel capsid expression strategy compared with other characterized tetraviruses. The capsid precursor protein is cleaved twice to generate three proteins. Two of the proteins, L (58.3 kDa) and S (6.8 kDa), are incorporated into the TaV virion. The third, non-structural protein, produced from the N terminus of the precursor protein, is up to 17 kDa in size and is of unknown function. The TaV capsid precursor protein sequence without the 17 kDa N-terminal region was modelled against the solved structure from Nudaurelia omega virus (N omega V) using SwissModel. The TaV model was very similar to the solved structure determined for subunit A of N omega V and had features that are conserved between tetraviruses and nodaviruses, including the positioning of the cleavage site between the L and S capsid proteins. The production of virus-like particles (VLPs) using the baculovirus expression system was used to analyse the capsid processing strategy employed by TaV. VLPs were formed in both the presence and absence of the 17 kDa N-terminal region of the capsid precursor. VLPs were not formed when the L and S regions were expressed from separate promoters, indicating that cleavage between the L and S capsid proteins was an essential part of TaV capsid assembly. Expression of the TaV 17 kDa protein in bacteria did not produce intracellular tubules similar to those formed by bacterial expression of the p17 protein from Helicoverpa armigera stunt virus.

Animals↗
PubMed1971

Virulence and pathogenesis of yellow fever virus serially passaged in cell culture.

Viscerotropic virulence of the Asibi strain of yellow fever virus (YFV) for monkeys has been known to be lost after serial passage in HeLa cell monolayers. This phenomenon was investigated in several other mammalian and insect tissue cell lines. Assay in monkeys of original seed virus and of virus after 7 and 11 passages in a porcine kidney cell line (PK) indicated essentially equal infectivity and mortality. Moreover, monkeys receiving the passaged virus exhibited more rapid onset of disease and death than animals infected with original seed virus. Histological changes in animals inoculated with passaged virus were identical to those in animals receiving the seed virus. Virus from later passages in PK cells was also lethal for approximately 50% of the monkeys; however, evidence for progressive attenuation was seen in these preparations. Similar results were obtained with a mosquito (Aedes aegypti) cell line. In contrast to results obtained in PK and mosquito cells, YFV became essentially avirulent (nonlethal and less infective) for monkeys after only seven passages in HeLa cell cultures.

Aedes↗